支气管肺泡灌洗液淋巴细胞亚群对肺部病变的诊断意义

目的 比较肺部感染,肺恶性肿瘤及间质性肺病患者支气管肺泡灌洗液细胞计数及淋巴细胞亚群的表达水平,评价其临床意义.方法 选取2017年5月至2019年12月收治的98例患者,其中肺部感染56例,肺恶性肿瘤18例,间质性肺疾病24例,检测肺泡灌洗液计数及淋巴细胞亚群水平.结果 肺部感染组中性粒细胞百分比及总NK细胞百分比显...     

多发性硬化症发作期外周血和脑脊液淋巴细胞亚群的分析

为分析多发性硬化症 (MS )患者发作期淋巴细胞亚群及给予甲基强的松龙 (MP )治疗后的变化 ,流式细胞仪测定 2 6例处于复发期MS患者外周血 (PB )和脑脊液 (CSF )及 8例MS患者予MP治疗后PB淋巴细胞CD3+ 、CD4 + 、CD8+ 、CD4 5RA+ 、CD4 + /CD4 5RA+ 、CD4 + /CD2 9+ 、CD19+ 、CD5 + /CD19+ 的百分率。结果发现MS患者PB中CD8+ 、CD4 5RA+ 和CD4 + /CD4 5RA+ 百分率降低 ,CD4 + /CD2 9+ 百分率和CD4 + /CD8+ 比值升高 ;CSF中CD3+ 、CD4 + 、CD4 + /CD2 9+ 百分率和CD4 + /CD8+ 比值高于PB ;淋巴细胞亚群与临床伤残程度和距此次发作的时间无关 ;MP治疗不影响PB淋巴细胞亚群变化。表明MS患者淋巴细胞通过血脑屏障有选择性 ,淋巴细胞亚群的变化在MS发病机制中起作用     

Flow Cytometric Analysis of Lymphocyte Activation in the Mixed Lymphocyte Response

Interleukin 2 receptor (IL2R) expression may be a useful parameter for assessing lymphocyte activation in the mixed lymphocyte response (MLR). However, the contribution of irradiated stimulator (S*) cells to the levels of IL2R⁺ cells recovered must first be defined. We have used a flow cytometric parameter termed the sip count to assess this potential contribution of S* cells. This parameter, which is the number of cells within a defined cell gate, is a reflection of the viable cell number per culture well, since (a) a constant number of cells were plated per well on day 0, (b) cells recovered from a well were resuspended in a constant volume, (c) the flow cytometer aspirated (sipped) a constant volume of cell suspension, and (d) nonviable cells were not included in the gate. Sip count assessment showed that only 5% of S* cells were recoverable by day 4 of culture; in contrast, 70% or more of unstimulated responder cells were recoverable. Sip counts of MLR cultures identified an increase in cell number beginning on day 5, reflecting DNA synthesis and cell division. We then used the sip count to assess changes in the levels of IL2R⁺ cells in MLR cultures. The number of IL2R⁺ cells continued to increase up to day 7, even though maximal DNA synthesis occurred on day 5. Further, dual color analysis revealed that the proportion of CD4 cells expressing IL2R was maximal on day 5, whereas the proportion of CD8 cells expressing IL2R continued to increase until day 9. These findings show that flow cytometry can be used to study lymphocyte activation by alloantigens.     

Cytochemical Analysis of Human Peripheral Blood Lymphocyte Subsets Defined by Monoclonal Antibodies

Human blood lymphocyte subpopulaiions, revealed by a panel of commercially available monoclonal antibodies by means of a resetting technique, were submitted to direct cyto-chemicat analysis and were shown to have distinguishing characteristics. T cells reactive with OKT3 antibody (T3⁺) displayed higher beta-glucuronidase and dot-like alpha-napnthyl acetate acid esterase (ANAE) activity than T3⁻ cells. Helper/inducer cells (T4⁺) were characterized by a high level of dot-like ANAE activity, whereas cytotoxic/suppressor cells (T8⁺) displayed selective naphthol-ASD-chloroacetate esterase activity. These results provide evidence for association of some cytoplasmic enzyme activities with the expression of membrane differentiation markers defined by monoclonal antibodies.     

Peripheral Blood Lymphocyte Subsets in Adolescents: a Longitudinal Analysis from the REACH Project

Flow cytometry analysis of lymphocyte subset markers was performed for a group of sexually active, human immunodeficiency virus (HIV)-negative adolescents over a 2-year period to establish normative data. Data were collected in the REACH Project (Reaching for Excellence in Adolescent Care and Health), a multicenter, longitudinal study of HIV-positive and high-risk HIV-negative adolescents. Two- and three-color flow cytometry data were collected every 6 months for these subjects. We determined the effects of gender, race, and age on the following lymphocyte subset markers: total CD4⁺ cells, CD4⁺ naïve cells, CD4⁺ memory cells, all CD8⁺ cells, CD8⁺ naïve cells, CD8⁺ memory cells, CD16⁺ natural killer cells, and CD19⁺ B cells. Gender was the demographic characteristic most frequently associated with differences in lymphocyte subset measures. Females had higher total CD4⁺ cell and CD4⁺ memory cells counts and lower CD16⁺ cell counts than males. Age was associated with higher CD4⁺ memory cell counts as well as higher CD8⁺ memory cell counts. For CD19⁺ cells, there was an interaction between age and gender, with males having significantly lower CD19⁺ cell counts with increasing age, whereas there was no age effect for females. Race and/or ethnicity was associated with differences in total CD8⁺ cell counts and CD8⁺ memory cell counts, although both of these associations involved an interaction with gender.     

Lymphocyte Subsets in the Blood

Removal of the largest single lymphoid organ, the spleen, leads to an increase in severe infections. To prevent this, transplantation of splenic fragments can be performed, which may, however, cause an increase in CD8⁺ lymphocytes in the blood of these patients. This is controversial since in the clinical situation it is often difficult to account for the different age of the patients, the time point after the operation and many other factors known to influence the number of lymphocyte subsets.
Using a well-defined animal model, B, T, CD4⁺, and CD8⁺ lymphocytes were determined preoperatively in adult rats. Then, either sham splenectomy, splenectomy, or splenic autotrans-plantation was performed and the animals were followed up for 15 months after the operation.
The surgical procedure itself, the site of blood sampling and ageing all influenced the number of lymphocyte subsets profoundly. Furthermore, giving the data as relative or absolute numbers leads to different results.
Splenectomy caused lymphocytosis, due to a significant increase in B and CD8⁺ lymphocytes, as did splenic autotransplantation, which indicates that the number of lymphocyte subsets in the blood should not be used to argue in favour of or against splenic autotransplantation.
This study demonstrates that the number of lymphocyte subsets in the blood is influenced by many factors and therefore should be determined in a highly standardized fashion.     

Age-Related Changes in Blood Lymphocyte Subsets of Saudi Arabian Healthy Children

The age-related changes in absolute and percentage values of lymphocyte subsets in the peripheral blood of healthy children of different ages (1 month to 13 years) were studied by flow cytometry. The absolute and percentage values for most lymphocyte subpopulations differed substantially with age. Comparisons among age groups from infants through adults revealed progressive declines in the absolute numbers of leukocytes, total lymphocytes, and T, B, and natural killer (NK) cells. The percentages of T cells increased with age. Within the T-lymphocyte population, the CD8⁺ subset increased but the CD4⁺ subset decreased, resulting in a declining CD4⁺/CD8⁺ ratio. The percentage of B cells declined, but that of NK cells remained unchanged. The percentage of HLA-DR⁺ T cells increased over time, but their number changed inconsistently. Our findings confirm and extend earlier reports on age-related changes in lymphocyte subpopulations. These data should be useful in the interpretation of disease-related changes, as well as therapy-dependent alterations, in lymphocyte subsets in children of different age groups.     

Aging and Lymphocyte Subsets in the Spleen and Peripheral Blood of the Sprague-Dawley Rat

This study was undertaken to determine the effects of aging on lymphocyte subsets in the peripheral blood and spleens of Sprague-Dawley rats. Rats aged 3, 13 and 26 months were used in the study. Analyses of dual labeled lymphocytes from the 26 month animals show decreases in the numbers of lymphocytes due to decreased cellularity (spleen) or reduced lymphocyte percentages within the total white blood cell population (peripheral blood). In the spleens and blood of the oldest rats, there were reduced numbers of Total T, T helper/ amplifier (T_h/a), virgin T_h, and natural killer (NK) cells. Other changes were observed in the spleen but not peripheral blood. The numbers of T cytotoxic/suppressor cells (T_c/s) B cells, “autoimmune” B cells and NK cells were reduced in the spleen but remained within normal limits in peripheral blood. The data show aging exerts different effects on the peripheral blood and splenic compartments of the immune system. These differences may have teleological significance in relation to immune responses to xenobiotics and neoplastic cells.     

Correlation between Sputum and Bronchoalveolar Lavage Fluid Cultures

A correlation study of the cultured bacteria from paired sputum and bronchoalveolar lavage fluid samples was performed. The rates of concordant culture-positive paired specimens that were isolated within 1 or 7 days were 93.7% and 96.5%, respectively, suggesting that the culture of readily collectable sputum specimens may result in useful microbiologic diagnosis.     

Identification and Phenotyping of Leukocytes in Bovine Bronchoalveolar Lavage Fluid

A method is proposed to identify leukocyte subpopulations in bovine bronchoalveolar lavage fluid by dual-laser flow cytometry. The technique uses several parameters, i.e., exclusion of highly autofluorescent alveolar macrophages and inclusion of leukocytes on the basis of labeling by specific antibodies and light scatter characteristics.     

T淋巴细胞在不同类型白血病患者外周血中的变化

目的通过对不同类型白血病患者外周血T淋巴细胞亚群(CD3+、CD4+、CD8+)的检测以评价不同类型白血病患者的细胞免疫功能。方法采用流式细胞术检测200例不同类型白血病患者和52例健康志愿者外周血T细胞亚群的水平。结果与正常对照组相比,急性白血病和慢性白血病患者的CD4+/CD8+比值都是下调的(P<0.05);急性白血病患者(包括急淋和急粒)的CD3+、CD8+细胞水平是上升的,且急淋患者还伴随着CD4+细胞的降低(P<0.05);慢性白血病患者的CD3+、CD4+细胞水平是下调的,其中慢淋患者的CD3+细胞下调更为显著,而慢粒患者则是以CD8+细胞增高为主(P<0.05)。结论不同类型白血病患者外周血T淋巴细胞均存在免疫异常,且具有不同异常方式,对指导治疗及疗效观察有一定的临床意义。     

Stability of Peripheral Blood Lymphocyte Count and E-Rosette Forming Lymphocytes in Healthy Adults

Highly reproducible peripheral blood lymphocyte (PBL) count and percent E-rosette forming lymphocyte (%E-RFL) assays were developed by modifying existing procedures. PBL count assay variation was reduced by using replicate electronic white blood cell (WBC) counting and 2,000 WBC differentials. The %E-RFL assay modifications reduced variation and include the use of: (a) a capillary buffy coat isolation procedure that recovered more than 85% of the PBL without the use of chemical separation media, (b) centrifugation temperatures of 20-22°C, and (c) toluidine blue staining that allowed enumeration of %E-RFL and not E-rosette forming cells, by exclusion of nonlymphoid cells. Day-to-day variation of PBL count and %E-RFL was defined by making serial determinations over a period of months. The data indicate that healthy adults maintain PBL counts and %E-RFL within narrow ranges that are specific for each individual.     

Flow Cytometric Analysis of In Vitro Proinflammatory Cytokine Secretion in Peripheral Blood from Multiple Sclerosis Patients

The cytokines, interferon- (IFN-), tumor necrosis factor- (TNF-rpar;, and interleukin-2 (IL-2) are important endogenous proinflammatory proteins and have been linked to disease activity in multiple sclerosis. In this study, we use flow cytometric methodology to compare the secretion of IFN-, IL-2, and TNF- from peripheral blood-derived T cells of multiple sclerosis patients to the secretion in healthy controls. The percentages of IFN-, IL-2, and TNF- secreting cells are not significantly different between multiple sclerosis patients and controls. However, the TNF- secreting CDS cell percentage is correlated with the IFN- and IL-2 secreting CD3 cell percentages in multiple sclerosis patients. In the controls, only the TNF- secreting CD3 cell percentage is correlated with IFN-. These findings show that correlated secretion of cytokines occurs in multiple sclerosis and suggest that concerted intercytokine interactions may play an important role in the disease.     

Stability of Peripheral Blood Lymphocyte Count and E-Rosette Forming Lymphocytes in Healthy Adults

Highly reproducible peripheral blood lymphocyte (PBL) count and percent E-rosette forming lymphocyte (%E-RFL) assays were developed by modifying existing procedures. PBL count assay variation was reduced by using replicate electronic white blood cell (WBC) counting and 2,000 WBC differentials. The %E-RFL assay modifications reduced variation and include the use of: (a) a capillary buffy coat isolation procedure that recovered more than 85% of the PBL without the use of chemical separation media, (b) centrifugation temperatures of 20–22°C, and (c) toluidine blue staining that allowed enumeration of %E-RFL and not E-rosette forming cells, by exclusion of nonlymphoid cells. Day-to-day variation of PBL count and %E-RFL was defined by making serial determinations over a period of months. The data indicate that healthy adults maintain PBL counts and %E-RFL within narrow ranges that are specific for each individual.     

IgG Subclasses in Bronchoalveolar Lavage Fluid from Patients with Asthma

We have measured Immunoglobulin G (IgG) subclasses in serum and bronchoalveolar lavage fluid (BALF) from 12 non-smoking patients with stable asthma and 9 non-smoking healthy volunteers to obtain information on their possible role in local immunological reactions. The quotients (concentration of IgG subclass in BALF)/(concentration of IgG subclass in serum) were calculated. In controls QIgG3 were lower than QIgGI, QIgG2 and QlgG4.
The IgG subclasses in BALF and epithelial lining fluid (ELF) from patients with asthma were significantly higher than in controls, mainly due to increased leakage from the blood. Again QIgG3 were lower than Q of other subclasses. In the analysis of local production of IgG, albumin or ceruloplasmin was used as reference protein. Several patients showed a local production or a preferential accumulation of one or more IgG subclasses.
We conclude that in healthy persons the IgG subclasses in ELF originate from the systemic circulation by passive permeation. In patients with asthma, the permeability of the respiratory membrane may be increased resulting in increased concentrations of subclasses in lung-lining fluid. In some patients with asthma, an additional local production of IgG subclasses occurs.     

Lymphocyte Subpopulations in the Cerebrospinal Fluid and Peripheral Blood in Patients with Multiple Sclerosis

The cerebrospinal fluid (CSF) and blood of patients with multiple sclerosis (MS) were studied with respect to the frequency of lymphocytes with intra-cellular immunoglobulins of different Ig classes as well as the relative frequency of B and T lymphocytes. An increased number of Ig-positive cells were found in CSF (mean, 0.52%) when compared with blood (mean, 0.18%). In CSF there was a striking dominance of IgG-positive cells, very few IgA-positive cells, and almost no IgM-positive cells. The distribution in blood was approximately normal. The ratios between x ⁻ and λ-positive cells in CSF were all outside the range in blood. In CSF there were fewer B cells (mean, 4.7%) and more T cells (mean, 74.2%) when compared with blood (mean, 11.5% and 61.8%, respectively). The values for MS blood were approximately the same as for normal controls. The increased number of IgG-containing cells in the CSF are in agreement with earlier studies, which showed a local immunoglobulin synthesis. The increased proportion of T lymphocytes in CSF of MS patients may indicate that these cells play a role in the pathogenesis of MS.     

Lymphocyte Subpopulations in the Cerebrospinal Fluid and Peripheral Blood in Patients with Multiple Sclerosis

The cerebrospinal fluid (CSF) and blood of patients with multiple sclerosis (MS) were studied with respect to the frequency of lymphocytes with intracellular immunoglobulins of different Ig classes as well as the relative frequency of B and T lymphocytes. An increased number of Ig-positive cells were found in CSF (mean, 0.52%) when compared with blood (mean, 0.18%). In CSF there was a striking dominance of IgG-positive cells, very few IgA-positive cells, and almost no IgM-positive cells. The distribution in blood was approximately normal. The ratios between χ- and λ-positive cells in CSF were all outside the range in blood. In CSF there were fewer B cells (mean, 4.7%) and more T cells (mean, 74.2%) when compared with blood (mean, 11.5% and 61.8%, respectively). The values for MS blood were approximately the same as for normal controls. The increased number of IgG-containing cells in the CSF are in agreement with earlier studies, which showed a local immunoglobulin synthesis. The increased proportion of T lymphocytes in CSF of MS patients may indicate that these cells play a role in the pathogenesis of MS.     

Flow Cytometric Analysis of CD133- and EpCAM-Positive Cells in the Peripheral Blood of Patients with Lung Cancer

Lung tumors are characterized by their high metastatic potential, which is the main cause of therapeutic failure. However, the exact cellular origin of metastasis remains unknown. Since the introduction of the cancer stem cell theory, lung cancer stem cells (LCSCs) have been thought to represent metastasis-founding cells. The current study aimed to evaluate whether LCSCs could be found in the circulation. Expression of the stem cell markers CD133 and EpCAM was confirmed in tumor and normal lung tissue by flow cytometry. Then, this technique was further used to investigate the expression of CD133 and EpCAM in the peripheral blood of 41 patients with primary lung cancer. Putative LCSCs (CD133⁺EpCAM⁺) were present in 6/7 tumor samples, and CD133⁺EpCAM⁺ cells were identified in the blood samples of 15 patients at a median level of 40/ml of blood. EpCAM⁺ cells were detected in 60 % of the patients, and the number of these cells was higher in patients with adenocarcinoma than patients with squamous cell carcinoma and was also higher in patients with less advanced disease. Moreover, the frequency of this subpopulation significantly correlated with the circulating level of SSEA-4⁺ cells. Additionally, CD133⁺EpCAM^? cells were found in 87 % of the patients, and the numbers of these cells were significantly higher in patients with distant metastases and correlated with disease stage. This study confirmed the presence of an LCSC subpopulation with a CD133⁺EpCAM⁺ phenotype in the tumors and blood of patients with lung cancer, and these results suggest an important role for CD133 and EpCAM in lung cancer progression and their potential application as novel biomarkers of the disease.